The occurrence and distribution of Xanthophyllomyces dendrorhous associated with Cyttaria hariotii parasitizing three Nothofagus species (N. dombeyi, N. antarctica and N. pumilio) in northwestern Patagonia (Argentina), as well as the factors that may affect this distribution were herein studied. Between 2000 and 2007, samples were obtained from 18 different locations. Based on physiological tests and morphological characteristics of sexual structures, 72 isolates were identified as X. dendrorhous. Representative strains were studied by MSP-PCR fingerprinting and sequence analysis of the ITS region. MSP-PCR fingerprints were similar for the newly isolated strains, and were also identical to the profiles of the strains previously found in this region. Patagonian strains appear to be a genetically uniform and distinct population, supporting the hypothesis that the association with different host species has determined genetically distinct X. dendrorhous populations worldwide. X. dendrorhous was recovered from N. dombeyi and N. antarctica. Approximately half the sampling sites and samples were positive for X. dendrorhous, but the isolation recovery rate was low. X. dendrorhous was absent in the early stages of ascostromata maturation, becoming more abundant in later stages. The present work represents a step forward in the understanding of the natural distribution and ecology of this biotechnologically relevant yeast.

Xanthophyllomyces dendrorhous (sexual stage Phaffia rhodozyma) is a basidiomycetous yeast that develops pink to red colonies, and has a set of unique features among yeast species. On the one hand, its main carotenoid pigment is astaxanthin, an economically important pigment that is absent in other yeasts (9). On the other hand, it couples the ability to produce this pigment with the ability to ferment simple sugars (17). This combination of characteristics is unique among yeast species. Original isolations of X. dendrorhous, which have served for most of the studies involving this species, were carried out in the 1960s by Phaff et al (17). These isolates were obtained from slime exudates of various broadleaved trees in different mountainous regions in the northern hemisphere, such as Japan and Canada. More recently, isolations of this species have been carried out in Italy (22), Germany (23) and Argentina (13). The isolations carried out in northwestern (NW) Patagonia, Argentina were the first to be reported in the southern hemisphere, and unlike all other previous isolations, they were not from sap flow, but rather from ascostroma of Cyttaria hariotii (13) parasitizing Nothofagus dombeyi. However, this study was performed in a single location and Nothofagus species, thus the widespread occurrence of X. dendrorhous in the Patagonian Nothofagus forests lacked confirmation. The Patagonian strains of X. dendrorhous bear genetic differences when compared to collection X. dendrorhous strains, yet their assignment to that species was supported by a high DNA homology based on DNA-DNA reassociation assays (13). The genetic differences within the species (intraspecific variability) have been hypothetically explained by geographic isolation and habitat specificity (13).C. hariotii is an ascomycetous fungus, which is endemic of the south hemisphere and exclusively parasites Nothofagus spp., the main tree genus of the Andean-Patagonian forests (2) and in particular of Nahuel Huapi National Park. Besides being very abundant, C. hariotii is one of the most widely distributed species of the genus Cyttaria. Its distribution coincides with that of the genus Nothofagus in South America, i.e. from around 33ºS latitude in central Chile to 56º in Tierra del Fuego (Argentina) (2). Five of the South American Nothofagus species are susceptible to the parasitic C. hariotii fungus: N. dombeyi, N. antarctica, N pumilio, N. betuloides, and N. nitida (5), of which only the first three are present at Nahuel Huapi National Park. The purpose of the research reported here was to study the distribution of X. dendrorhous in Patagonian Nothofagus forests (NW Patagonia, Argentina), especially concerning its occurrence and association to C. hariotii from the three main species that grow at Nahuel Huapi National Park (N. dombeyi, N. antarctica and N. pumilio). Methodology was based on C. hariotii sample collection, yeast isolation, phenotypic characterization of the isolated yeasts and molecular characterization of suspected X. dendrorhous isolates. Factors like maturity of ascostromata and presence of insects/larvae that may affect the distribution of this yeast were also analyzed.

Figure 1. Geographic location of the sampling sites (Nahuel Huapi National Park, NW Patagonia, Argentina). Black circles correspond to X. dendrorhous positive sites and light grey circles indicate where X. dendrorhous was not found. Grey areas represent waterbodies. Arrows help associate sites 3 and 14 to their respective site number. Site 7 is not included because it was located near the city of Bolson (outside the mapped area).

Table 1. Additional information for surveyed sampling sites (a)Please use this code number to locate site on the map in Figure 1. Note that site 1 was sampled in two different years, hence the letters a and b follow the site code number.

Sampling was carried out in the months of November-December (spring), between 2000 and 2007. One to 10 samples of C. hariotii, comprising one to 11 ascostromata each, were aseptically collected at each location. Samples were stored in refrigerated sterile flasks until processing upon arrival at the laboratory (< 48 h). For each sample, the number of ascostromata and the fresh weight of the samples were registered. Three ascostromata maturity stages were considered as follows, (i) immature: ascostroma covered by a tough, elastic skin or cortical layer, which becomes membranous and tightly stretched; (ii) mature: ruptured cortical layer exposing mouths of apothecia; (iii) overmature: ascostroma has lost turgidity, cortex grows old, and the distinct odor of alcoholic fermentation may be perceived. Finally, ascostromata were checked for presence of insects/larvae. Geographic location and altitude of each site were registered using a GPS (Garmin, Legend, USA). Survey of yeasts present in the samples was carried out as described by Libkind et al. (13), with minor modifications. Ascostromata were cut into cubes (approximately 2 x 2 cm), placed in bags with sterile distilled water (1:1 w/v), and crushed manually inside the bags. The bag content was then transferred to Erlenmeyer flasks and shaken at 20 °C for 30 min at 300 rpm. Aliquots of 100 µl of the extracts (diluted so as to obtain no more than 300 colony-forming units per plate) were inoculated on YPD agar (g/l: yeast extract 10, peptone 20, glucose 20, agar 15). Culture medium was adjusted to pH 4.5 - 5 and supplemented with 200 mg/l chloramphenicol. Incubation temperature was 15-18 °C. Depending on the methodology used, isolations obtained could correspond both to the surface or the internal portion of the stromata. Pigmented colonies that appeared on the plates were transferred to fresh YPD agar plates and were tested for production of amyloid compounds and ability to ferment glucose (25). Sexual stage formation was assessed as described by Kucsera et al. (11). Twenty-nine representative isolates were stored both by refrigeration (4 °C) on YMA (g/l, yeast extract 3; malt extract 3; peptone 5; dextrose 10; agar 20) and by deep-freezing (-180 °C) in liquid nitrogen.

Molecular characterization

X. dendrorhous isolates were characterized by the minisatellite primed-PCR technique (MSP-PCR) and then subjected to cycle sequencing of the ITS rDNA region. Pigmented isolates other than X. dendrorhous were also characterized by MSP-PCR, but sequencing was performed for the D1/D2 domains of the 26S rRNA gene. The DNA extraction protocol, MSP-PCR and electrophoresis conditions, and gel image analysis procedures were those reported in Libkind et al. (14). The primer employed was the core sequence of the M13 phage (5´-GAGGGTGGCGG- TTCT-3´). For sequence analysis, rDNA was amplified using the forward primer ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´) and the reverse primer ITS4 (5´-TCCTCCGCTTATTGATATGC-3´), as described in Libkind et al. (13). Alignments were made with BioEdit v7.0.9.0 (8), and visually corrected. Phylogenetic relationships were estimated using the MEGA program version 4.0.2 (19); the phylogenetic tree was constructed using the neighbor joining (NJ) algorithm, and bootstrap values calculated from 1000 replicate runs. The Kimura two-parameter model (10) was used to estimate evolutionary distance. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). Sequences available in the GenBank database were used for comparative purposes.

RESULTS

Over 100 orange-pigmented yeast isolates were recovered from 56 samples of C. hariotii collected from 18 different locations at Nahuel Huapi National Park (Figure 1). Seventy-two of these isolates were considered X. dendrorhous-like, for their ability to produce amyloid compounds and ferment glucose. Patagonian isolates produced typical sexual structures of X. dendrorhous, confirming that they belong to X. dendrorhous and suggesting these Patagonian strains constitute a teleomorphic population of fungi. As depicted in Figure 2, DNA profiles obtained from MSP-PCR fingerprinting were highly similar for all isolated strains. Isolates were identified as X. dendrorhous, based on ITS sequence analysis of representative strains . In agreement with the MSP-PCR results, all sequences obtained here were 100 % identical to those Patagonian strains previously sequenced (for example DQ661028) (Fig. 3).

Figure 3. Phylogenetic relationships among X. dendrorhous strains inferred by ITS sequence analysis using the Neighbor-Joining method (18). Bootstrap analysis was performed with 1000 replicates (4). The evolutionary distances were computed using the Kimura 2-parameter method (10), and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4 (19). Sequences of CRUB strains lacking Genebank accession numbers were obtained in this study.

Other pigmented yeast species found in C. hariotii were Rhodotorula mucilaginosa, Rhodotorula colostri, Cystofilobasidium infirmominiatum, Cystofilobasidium capitatum, Cystofilobasidium macerans, and the yeast-like fungus Aureobasidium pullulans. Cystofilobasidium yeasts were the most frequently isolated pigmented species, and frequently occurred simultaneously with X. dendrorhous in the same sample of C. hariotii. Considering that Cystofilobasidium yeasts grow in orange colonies, produce amyloid compounds and may weakly ferment glucose, colonies of this yeast may potentially be misclassified as X. dendrorhous-like.X. dendrorhous was found in 45% of the samples, corresponding to 10 sampling sites (Table 2). Samples of C. hariotii were taken from three Nothofagus species (N. dombeyi, N. antarctica, and N. pumilio), but X. dendrorhous was only recovered from N. dombeyi and N. antarctica (Table 2). Ascostromata maturity influenced the X. dendrorhous recovery rate. Immature ascostromata were mostly negative for X. dendrorhous, while approximately 75 % of samples corresponding to mature and over-mature ascostromata were positive (Table 3).

Table 3. Sample and X. dendrorhous isolate distribution for different maturity stages of C. hariotii ascostromata(a) Calculated over the total number of isolates for which maturity data was available (n = 51).

DISCUSSION

DNA profiles obtained from MSP-PCR fingerprinting were identical to those of the strains previously found in this region, and thus differed from those obtained from other regions around the world (13, 15). Judging by the ITS sequences, Patagonian strains appear to be a genetically uniform and distinct population considering that the new isolates herein obtained shared identical ITS sequences to those of previous studies. As depicted in Figure 3, three main clades can be distinguished. Patagonian strains form a homogenous group and constitute a sister clade of the main clade of north hemisphere strains which are associated to exudates of Betulaceae trees from different geographical areas. The case of the type strain of P. rhodozyma CBS 5905T isolated from Fagaceae is still not clear, as previously addressed (13). The third clade includes only two strains from Cornaceae trees in Japan and is the least represented and more distantly related group. When considering the ecology of the north hemisphere strains of X. dendrorhous and the ephemeral nature of Cyttaria stromata, it becomes highly probable that the Patagonian population is actually associated to other tree-related substrates such as soil or leaves. However, very few cases of X. dendrorhous isolations from phylloplane have been reported so far (16, 24) and none from soil, which can be explained by a very low abundance maybe limited to a few spores. Due to the more favorable nutritional conditions of the stromata, opportunistic colonization by X. dendrorhous takes place when their fructification occurs in late spring. Similarly, X. dendrorhous-colonized exudates in the north hemisphere appear only in spring, providing this yeast the opportunity to propagate. Based on the idea that the Patagonian strains are associated to the Nothofagus host, we previously proposed a host specificity model which suggested that the different yeast lineages (clades formed in Figure 3) colonize different tree species, and attribute genetic variation (intraspecific variation) to host specificity (13). The new information gathered in the present work supports this hypothesis, considering that the new isolates herein obtained from N. antarctica group together with those of the same genus. It remains to be studied if molecular markers with higher resolution could detect genetic subpopulations of Patagonian X. dendrorhous and if they are related to different Nothofagus species. This would help prove if the host specificity model can also be applied at host species level as well as at host family level, as already seen. Several reports have shown the simultaneous presence of X. dendrorhous and Cystofilobasidium spp. (typically C. infirmominiatum, C. macerans and/or C. capitatum) in birch exudates (6, 7, 17, 21, 22). Apparently, when X. dendrorhous is found, Cystofilobasidium yeasts are also typically present. The opposite is not necessarily so, given that the natural distribution of Cystofilobasidium is broader than that of X. dendrorhous. Both genera belong to the monophyletic order Cystofilobasidiales, and thus share several physiological characteristics such as carotenoid production (3). Further studies are needed to understand the apparent niche overlap between X. dendrorhous and some Cystofilobasidium yeasts. Despite the fact that about half of the total samples were positive for X. dendrorhous, positive samples yielded a low number of isolates, generally just one or two (only three samples yielded more than three isolates), which suggests that optimizing isolation procedures may be necessary to increase isolation recovery rates. It is clear from recent research that habitats and distribution of X. dendrorhous are broader than originally suspected (13, 24). The present study, in particular, is the first report for X. dendrorhous growing on a Nothofagus species other than N. dombeyi, i.e. N. antarctica, and also shows that the occurrence of this yeast in C. hariotii is a general phenomenon in Nothofagus forests of NW Patagonia. We failed to find X. dendrorhous in C. hariotii from N. pumilio, likely due to the low sample size. Within Nahuel Huapi National Park, N. pumilio is usually restricted to altitudes above 1000 m.a.s.l. (2), and such altitudes were rarely covered in this study. However, in a previous work, we suggested that X. dendrorhous that had been isolated from lake water was originally from C. hariotii growing on N. pumilio surrounding the lake (15). Moreover, in a recent study dealing with the biodiversity of phylloplane fungi of N. pumilio at Nahuel Huapi National Park, a X. dendrorhous isolate was obtained (16). This result is in agreement with the proposal of X. dendrorhous as an epiphytic yeast (21, 24). In a previous study we found, for a single location, that the proportion of X. dendrorhous isolates was higher in mature ascostromata than in immature ascostromata (13); however, overmature ascostromata had never been studied. In the present study, more locations throughout Nahuel Huapi National Park were studied, and the great number of X. dendrorhous isolates recovered from mature ascostromata supported the previous observation. In addition, we observed that overmature ascostromata had a higher proportion of multiple (two or more) X. dendrorhous isolates than mature ascostromata. The increased isolate recovery rate for late maturity stages is probably related to the elevated sugar concentration in these stages (12). When mature, the fruiting bodies of C. hariotii have a sugar content of almost 10 % (mainly D-glucose, fructose, and sucrose) and contain polyols such as glycerol, D-mannitol, and D-arabinitol. Polyols are known to be key compounds for the development of the sexual cycle in X. dendrorhous (11). It is clear that the nature and ecology of the sap-flows in the northern hemisphere and Cyttaria ascostromata in the southern hemisphere are quite different. However, both these substrates harbor X. dendrorhous. Thus, some careful analogies may be drawn between them. In both cases, X. dendrorhous is absent in the early stages of microbial colonization, and becomes more abundant in the later stages of colonization (6, 7, 21). However, in sap-flows, X. dendrorhous becomes one of the dominant species in the late phases of colonization (6, 7, 22), whereas in C. hariotii, X. dendrorhous does not outnumber sympatric ascomycetous fermenting yeasts frequently found in overmature ascostromata such as Saccharomyces, Hanseniaspora, Pichia, Candida, and Zygosaccharomyces (1, 20). The second interesting analogy concerns the role of insects as vectors for X. dendrorhous. It has been suggested that insects are probably crucial as vectors for yeasts between sap-flows of different trees (21). The same is probably true for X. dendrorhous in C. hariotii. In the present work, it was not observed that C. hariotii ascostromata harboring insects/larvae had higher X. dendrorhous infection rates than those that did not. In summary, X. dendrorhous was recovered in very different and distant locations throughout the sampling area, not only from C. hariotii infecting N. dombeyi, as previously documented, but also from N. antarctica. The Patagonian strains formed a genetically distinct population, supporting our previously suggested hypothesis that the association with different host species has determined genetically distinct X. dendrorhous populations worldwide. The abundance of this yeast increased with the maturity stages of the stromata and arguments were provided in favor of an epiphytic nature of this yeast when stromata (or exudates from the north hemisphere) are absent. Future studies will focus on the development of selective isolation media to increase the isolation recovery rate of X. dendrorhous, as well as on the study of molecular markers with better discriminatory power than those of the ITS region.

Acknowledgements: This work was funded by Project B143 of the Universidad Nacional del Comahue, granted to M.V.B.; Project PICT 1745 of the Agencia Nacional de Promoción Científica y Tecnológica (Argentina), granted to D.L.; and Project PTDC/BIA-BDE/73566/2006 of the Fundação para a Ciência e a Tecnologia (Portugal), granted to J.P.S. We would like to thank the authorities of Parques Nacionales (Argentina), for authorizing sample collection within Nahuel Huapi National Park. We appreciate the contribution of Amalia Denegri in map design. We are thankful to two anonymous reviewers for their comments that largely improved the final manuscript.